A phylogenetic study on the activation mechanism of MC5R by the Pituitary Hormone ACTH

A phylogenetic study on the activation mechanism of MC5R by the Pituitary Hormone ACTH

HFRW analog interactions with srMC5R + esMRAP1

HFRW analogs with cMC5R + cMRAP1

Overall Conclusions

For srMC5R, it appeared that the order of importance for positions in the HFRW motif were: W=R>F=H

The pattern here was similar to that of srMC5R. However, the emphasis of position importance differs.

For the HFRW motif to interact with cMC5R, the amino acid position hierarchy is as follows: W>>R>F=H

This effect was similar to the effects with rtMC2R in another study. This could be due to the interactions between the R and W positions with srMC5R, in which those interactions facilitate tighter binding and perpetuate the activation of the receptor.

Hypothesis

• Based on the observations from two different species, it would appear that interaction between MC5R and MRAP1 is species specific.
• srMC5R activation does not involve exposure of a site on the receptor that could be classified as a KKRRP docking site; rather, the interaction with MRAP1 appears to lead to an enhancement of activation.

Cartoon of ACTH(1-24) binding at MC2R

The interaction between tryptophan (W) and cMC5R proved to be more critical for full activation than the other three amino acids.

For this study, it was hypothesized that, due to the common origin between mc2r and mc5r, MC5R would follow the two step binding mechanism. It was hypothesized that, like MC2R, it would require both the KKRRP motif and HFRW motif for full activation. It was also determined which amino acid positions in the HFRW motif were the most important. Lastly, it was tested whether the removal of glycines in the GKPVG motif of ACTH(1-24) or the removal of KPV in the motif prevented the binding of HFRW and KKRRP and thus blocked activation.

KKRRP analog interactions with srMC5R + esMRAP1

KKRRP analog interactions with cMC5R + cMRAP1

The substitutions in the KKRRP analog, however, did not have a significant effect on stimulation.

• The analysis of cMC5R co-expressed with cMRAP1 further underscored the importance of the HFRW motif for the activation of this receptor.
• The importance of the tryptophan (W) position was the same as seen for cMC2R in another study

The drops in sensitivity for cMC5R when stimulated with the KKRRP analogs were statistically significant from wild-type ACTH(1-24).

This effect differs from MC2R, in which both motifs are critical for full activation. Thus, it appears that srMC5R only requires the HFRW motif for activation.

This differs from srMC5R, but remains consistent with MC2R. In this study, cMC5R appears to require the KKRRP motif to some degree, though the HFRW motif does appear to be more critical for activation.

Yet, the A5 analog for the KKRRP motif fully eliminated activation. It is possible that the introduction of 5 alanine residues in sequence led to the formation of an alpha helix.

The alpha helix distorts the shape of hACTH(1-24) and prevents it from binding to the receptor at both the HFRW and the KKRRP sites.

As with the srMC5R interaction with the A5 analog, cMC5R activation was entirely wiped out, likely due to that formation of the alpha helix in the peptide structure.

• It appears that the KKRRP motif is needed to achieve full activation of cMC5R
• Hence, cMC5R does have pharmacological properties that are similar to cMC5R that would be consistent with the two-step model for activation

What are the melanocortin peptides?

MC2R + MRAP1

Pharmacology studies have shown that MC2R in bony vertebrates requires the co-expression of an accessory protein, MRAP1.

There are five melanocortin peptides: ACTH, alpha-MSH, βMSH, γMSH, delta-MSH. All are derived from the precursor protein proopiomelanocortin, or POMC.

Truncated hACTH(1-24) analog interactions with cMC5R + cMRAP1

Truncated hACTH(1-24) analog interactions with srMC5R + esMRAP1

Bringing it all together...

MRAP1 is a single pass transmembrane protein that interacts with MC2R to aid in translocation from the endoplasmic reticulum to the plasma membrane.

The removal of residues to form hACTH(1-21) and hACTH(1-22) had a greater effect on cMC5R activation than srMC5R activation.

The truncated forms of hACTH(1-24) were tested to see if there was spatial interference based on the two-step binding mechanism for activating MC2R orthologs.

• These observations raise the possibility that other bony vertebrate MC5R orthologs may have similar pharmacological features comparable to their MC2R paralogs

The complex of MC2R + MRAP1 is required at the plasma membrane for proper binding of ACTH in order for the target cell to be activated.

This could be because cMC5R appears to need the KKRRP motif to a certain degree for activation.

The melanocortin peptide of interest in this study is ACTH; POMC undergoes extensive, tissue specific, posttranslational modifications that leads to ACTH being the major end product in corticotropic cells of the anterior pituitary in mammals.

hACTH(1-21) had no statistically significant difference in activation, but hACTH(1-22) only showed activation at concentrations much higher than those seen in an organism.

Given the co-evolution of MC2R and MC5R, it's predicted that the activation of MC5R be influenced by interaction with MRAP1.

The removal of residues would physically inhibit the two-step mechanism of ACTH. It would constrain the peptide so the KKRRP and HFRW motifs could not bind in their pockets properly.

It appears that the removal of the glycines in hACTH(1-22) constrained the secondary structure of the analog and interfered with activation.

As srMC5R does not appear to require the KKRRP motif for activation, it makes sense that it is not as inhibited by the residue removals as cMC5R.

cMC5R + MRAP1

srMC5R + MRAP1

Binding mechanism of MC2R

In a chicken (c) adrenal gland, mRNAs for MC2R, MC5R, and MRAP1 have been detected.

Recent studies have shown that the interrenal cells of the red stingray contain mRNAs for MC2R and MC5R.

MC2R is the only melanocortin receptor that cannot bind to any MSH-sized peptides. This difference is likely due to the two-step binding mechanism and the influence of MRAP1. ACTH(1-24) contains the HFRW motif which MC2R requires for activation; in fact, all the melanocortin peptides have this motif. However, only ACTH(1-24) has the KKRRP motif, which is also required for MC2R activation across bony vertebrates. For MC2R, the KKRRP motif binds first, which then exposes a docking site for the HFRW motif. Only then can the cell begin a cascade for cortisol production.

The melanocortin receptors belong to the family of G protein-coupled receptors. They are activated by the melanocortin peptides and, in the case of MC2R, help an organism recover from a chronic stress event. The receptor of interest in this study, MC5R, originated on the same chromosome as MC2R. Thus, it is plausible that MC5R and MC2R share common pharmacological properties. The property of interest in this study was the binding mechanism of ACTH to MC2R, and whether that applied to MC5R.

Co-expression of srMC5R with MRAP 1 has also been shown to increase sensitivity to stimulation by ACTH(1-24) 100 fold.

Co-expression of cMC5R with cMRAP1 has been shown to increase sensitivity to stimulation by ACTH(1-24) 1000 fold.

Results

srMC5R + esMRAP1

Acknowledgements

Abstract

• When the receptor was stimulated with the AFRW analog, there was a decrease in receptor sensitivity of 2 orders of magnitude (p=0.02)
• When the receptor was stimulated with the HARW analog, there was a drop in receptor sensitivity of 3 orders of magnitude (p=0.01)
• When the receptor was stimulated with the A4 analog, there was no stimulation
• Stimulation with the HFAW and HFRA analogs resulted in virtually no activation
• Stimulation with the AARRP analog did not lead to a statistically significant drop in sensitivity (p=0.49)
• Similarly, when the receptor was stimulated with the KKAAA analog, there was no statistical difference in receptor sensitivity (p=0.38)
• However, stimulation with the A5 analog led to very minimal receptor activation
• Stimulation with hACTH(1-21), a truncated form of hACTH(1-24), led to activation that was not statistically significant from the positive control (p=0.24)
• Stimulation with hACTH(1-22) eliminated activation

Photo obtained from http://www.differencebetween.net/science/nature/animals-nature/difference-between-manta-ray-and-stingray/

I would like to thank Dr. Robert M. Dores for guiding me in this project and in writing this thesis. Without his help, I would not have been able to investigate this topic. I would also like to thank the Partners in Scholarship grant for providing funds to purchase supplies for this project.

cMC5R + cMRAP1

• Stimulation with the AFRW analog decreased receptor sensitivity by 2 orders of magnitude (p=0.05)
• When the receptor was stimulated with HARW, there was a drop in sensitivity of 3 orders of magnitude (p=0.05)
• When the receptor was stimulated with the HFAW analog, there was a significnat drop in sensitivity (p<0.001)
• Upon stimulation with the HFRA analog, there was virtually no receptor activation at all
• Stimulation with the A4 analog yielded the same effect
• When the receptor was stimulated with the AARRP analog, there was not a statistically significant drop in sensitivity (p=0.11)
• Stimulation with the KKAAA analog did result in a significant decrease in receptor sensitivity (p=0.05)
• Stimulation with the A5 analog also significantly affected receptor sensitivity (p=0.001)
• Both the truncated forms of hACTH(1-24) resulted in significant drops in sensitivity. hACTH(1-21) led to a drop in 2 orders of magnitude (p=0.002) as did hACTH(1-22) (p=0.02).

Figure 4: KKRRP analogs for cMC5R with cMRAP1

Figure 1: HFRW analogs for srMC5R with esMRAP1

Photo obtained from https://www.pinterest.com/pin/294352525626229683/

Table 1: ACTH(1-24) analogs used in this study

Figure 3: HFRW analogs for cMC5R with cMRAP1

Figure 2: KKRRP analogs for srMC5R with esMRAP1

Wild type human ACTH(1-24) SYSMEHFRWGKPVGKKRRPVKVYP

HFRW analog H to Alanine SYSMEAFRWGKPVGKKRRPVKVYP

HFRW analog F to Alanine SYSMEHARWGKPVGKKRRPVKVYP

HFRW analog R to Alanine SYSMEHFAWGKPVGKKRRPVKVYP

HFRW analog W to Alanine SYSMEHFRAGKPVGKKRRPVKVYP

HFRW analog A4 SYSMEAAAAGKPVGKKRRPVKVYP

ACTH(1-22) SYSMEHFRWKPVKKRRPVKVYP

ACTH(1-21) SYSMEHFRWGGKKRRPVKVYP

KKRRP analog KK to Alanine SYSMEHFRWGKPVGAARRPVKVYP

KKRRP analog RRP to Alanine SYSMEHFRWGKPVGKKAAAVKVYP

KKRRP analog A5 SYSMEHFRWGKPVGAAAAAVKVYP